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ISOLATED MITOCHONDRIA INFUSION MITIGATES ISCHEMIA-REPERFUSION INJURY OF THE LIVER IN RATS Han-Chen Lin,* Shin-Yun Liu,* Hong-Shiee Lai,† and I-Rue Lai*† *Department of Anatomy and Cell Biology, Medical College, National Taiwan University, and † Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan Received 13 Aug 2012; first review completed 29 Aug 2012; accepted in final form 13 Dec 2012 ABSTRACT—A recent study showed that the injection of mitochondria isolated from a nonischemic region mitigated myocardial injury. We tested the protective effects of infusing isolated mitochondria on the reperfusion injury in the liver of rats. A partial liver ischemia-reperfusion (I/R) model in male Wistar rats was used. At the 45th minute of liver ischemia, the recipient’s spleen was infused with vehicle (I/R-vehicle group) or vehicle containing isolated mitochondria (7.7  106 T 1.5  106/mL, I/R-mito group). After a 240-min reperfusion, the serum and livers were collected to assess tissue injury. Our results show that the elevation of serum alanine aminotransferase (414.3 T 67.1 vs. 208.8 T 30.2 U/L), the necrosis of hepatocytes on hematoxylin-eosin staining, increase in positive counts in TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining (59.5% T 4.4% vs. 24.6% T 9.1%), the expression of cytosolic cytochrome c, cleaved caspase 9, and 4-hydroxynonenal were all reduced in the I/R-mito group, compared with the I/R-vehicle group. The membrane potential of the isolated mitochondria measured by JC-1 fluorescence remained high, and the infused mitochondria were distributed in the liver parenchyma at 240 min after reperfusion. These results demonstrate that an intrasplenic infusion of viable mitochondria isolated from the donor before reperfusion significantly reduced I/R injury in the liver. KEYWORDS—Isolated mitochondria, membrane potential, reperfusion injury, liver ABBREVIATIONS—ALT V alanine aminotransferase; CCCP V carbonylcyanide m-chlorophenylhydrazone; I/R V ischemia-reperfusion; JC-1 V 5,5¶,6,6¶-tetrachloro-1,1¶,3,3¶-tetraethylbenzimidazolyl-carbocyanine iodide; $=m V membrane potential of the isolated mitochondria

INTRODUCTION

Another attractive alternative to cell transplantation is the replacement of damaged mitochondria during ischemia. McCully et al. (9) showed that the injection of isolated, functional mitochondria into the ischemic myocardium could enhance functional recovery of myocardium and decrease the infarct zone. The effects of mitochondria transfusion on reperfusion injury have not been examined in other organs. In this study, we tested the hypothesis that infusion of the isolated mitochondria could mitigate reperfusion injury in a rat model of hepatic ischemia.

Reperfusion injury is a clinically important issue in liver transplantation and inflow-controlled hepatectomy. Among the complex intracellular and extracellular signaling pathways involved in reperfusion injury, mitochondria have been found to be the key target. A wide array of functional alterations in mitochondria develops during reperfusion injury of liver (1), including the excessive generation of reactive oxygen species (ROS) and intracellular Ca2+ overload, which result in mitochondrial permeability transition (MPT) (2) and subsequent cytochrome c releaseYrelated cell death (3, 4). Attempts to mitigate mitochondrial insults and reperfusion injury include the pharmacological targeting of MPT, the modulation of reperfusion procedures, and cell-based therapy. NIM811, a selective MPT inhibitor, was shown to reduce reperfusion insults after liver transplantation (5). In our previous study, we showed a modulation of the reperfusion maneuver (ischemic postconditioning) mitigates hepatic reperfusion injury through the inhibition of MPT (6). Recently, researchers reported that systemically administered adipose-derived mesenchymal stem cells alleviate hepatic reperfusion injury (7) and promote liver regeneration (8).

MATERIALS AND METHODS Animal care and preparation Male Wistar rats weighing 200 to 250 g were used. All animal experiments and animal care were performed in accordance with the BGuides for the Care and Use of Laboratory Animals[ (published by the National Academy Press, Washington, DC, 1996). All of the protocols used in this study were approved by the Laboratory Animal Care Committee of the National Taiwan University College of Medicine.

General surgical procedures Rats were anesthetized with sodium pentobarbital (35 mg I kgj1, i.p.). The trachea was intubated to keep airway patent, and the right jugular vein was catheterized for infusion of saline with a rate of 20 I kgj1 I hj1. The carotid artery was catheterized for measurement of blood pressure and blood sampling. The mean arterial blood pressure was recorded on a Gould polygraph (Quincy, Mass). Body temperature was maintained at 36.5-C to 37.5-C using a heating pad.

Address reprint requests to I-Rue Lai, MD, PhD, Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Rd, Taipei, Taiwan. E-mail: [email protected] This work was originated at the Medical College of National Taiwan University and was supported by research grant from National Taiwan University Hospital, NTUH 100S1553. There is no conflict of interest in this work for every author listed. DOI: 10.1097/SHK.0b013e318283035f Copyright Ó 2013 by the Shock Society

Induction of hepatic ischemia-reperfusion injury After a 30-min stabilization period following the surgery, a modified rat model of partial hepatic ischemia-reperfusion (I/R) was performed (10). The portal triad was exposed. A partial hepatic I/R injury was produced by placing an atraumatic microvascular clip cephalad on the branches of the right and caudate lobes, thus occluding the hepatic artery, portal vein, and bile duct of the left and median lobe for 45 min (ischemia period); the clip was then 304

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SHOCK MARCH 2013 removed, and the area was allowed to reperfuse for 240 min. The belly of the animal was closed temporarily in the ischemic and reperfusion period except when accessing the spleen.

Isolation of rat liver mitochondria Mitochondria were isolated by standard differential centrifugation (11). The fresh liver was removed via a laparotomy after the rat was anesthetized. The tissue was homogenized in suspension buffer (0.5 M HEPES-KOH, 1 M KCl, 1 M MgCl2, 0.5 M EGTA, 0.5 M EDTA, protease cocktail [Sigma, St. Louis, Mo], phosphatase inhibitor I [Sigma], phosphatase inhibitor II [Sigma], 0.25 M sucrose, pH 7.6) by 25 strokes with a Dounce homogenizer at 4-C. The homogenate was centrifuged for 10 min at 750 g, the pellet was discarded, and the supernatant was recentrifuged for 20 min at 10,000g to isolate the mitochondria. The isolated mitochondria were stained with MitoTracker Orange CMTMRos (5 2mol/L; Invitrogen, Carlsbad, Calif) to determine their number (9). Briefly, 1 2L of labeled mitochondria were spotted onto slides and counted using a confocal microscope (Zeiss, Germany). The mitochondrial number is determined at low (10) magnification covering the entire specimen area. The purity of the isolated mitochondria was examined by detecting the mitochondrial marker (COX IV) expression in the lysate by Western blot analysis. For our experiment, three donor rats were used to harvest the isolated mitochondria. To identify the intactness of the isolated mitochondria, flow cytometry analysis of the mitochondrial membrane potential ($=m) was performed on the isolated mitochondria stained with JC-1 (5¶,6,6¶-tetrachloro-1,1¶,3,3¶tetraethylbenzi-midazolylcarbocyanine iodide). JC-1 is a lipophilic, cationic dye that can selectively enter mitochondria and undergoes a reversible change in fluorescence emission according to the$=m. Healthy cells with high $=m will form JC-1 aggregates and fluoresce red, whereas apoptotic or unhealthy cells with a low$=m will contain monomeric JC-1 and fluoresce green (12, 13). The isolated mitochondria (0.3 mg of protein) were incubated for 5 min at room temperature in the dark, in the presence of 1 2m JC-1( MitoProbe JC-1 Assay Kit, MP 34152; Molecular Probes, Eugene, Ore) as previously described (14). The suspensions were analyzed immediately by flow cytometry, using a FACSCalibur flow cytometer (BD Bioscience, San Jose, Calif). The change in $=m was reported by the ratio of red to green fluorescence. As a positive control, some of the isolated mitochondria were treated with the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP: 50 mM) to completely deplete $=m. The results of the JC-1 fluorescence were analyzed using Cellquest software (BD Biosciences, San Jose, Calif), and the red/green fluorescence emission of particles was recorded as a dot plot. The data were expressed as the mean T SEM, and at least three separate experiments were performed in each group.

Biodistribution of transplanted mitochondria in liver For anatomical and functional linkage, we observed the biodistribution of transplanted mitochondria in the liver. The isolated mitochondrial pellet was incubated with 1 2L MitoTracker Orange CMTMRos (Invitrogen) at 37-C for 30 min for later use. A midline laparotomy was performed on the anesthetized, recipient rat. The tracker-marked mitochondria (0.1 mL) were injected into the spleen. After 45-min ischemia and 240-min reperfusion, the liver was harvested and frozensectioned. The biodistribution of the injected mitochondria in the ischemic and nonischemic lobes was observed under fluoroscopy (for MitoTracker Orange CMTMRos: excitation 554 nm, emission 576 nm). In a preliminary test, MitoTracker only was infused via the spleen. No staining of MitoTracker was detected 240 min after the laparotomy, indicating that the native hepatocytes were not labeled by the MitoTracker.

Experiment protocol A partial liver I/R was used. The experimental protocol is depicted as below. The sham group received a laparotomy only. The sham-mito group received a laparotomy and a splenic injection of isolated mitochondria (7.7  106 T 1.5  106/mL) after 45 min of liver ischemia. The I/R-vehicle group received liver I/R injury and a splenic injection of MitoTracker dye only after the 43rd minute of liver ischemia. The I/R-mito groups received a splenic injection of isolated mitochondria (7.7  106 T 1.5  106/mL) after 45 min of liver ischemia. The injection site was at the subcapsular region of the spleen poles, and the injected volume was 100 2L. After 240-min reperfusion, blood and liver samples were harvested for further measurement.

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Liver histology Liver samples were harvested and fixed in 8% paraformaldehyde for 2 days and embedded in paraffin. The paraffin-embedded blocks were sectioned into 5-2m sections and stained with hematoxylin and eosin. The morphometric assessment of the liver injury was reviewed by a pathologist who was blinded to the grouping. The grading of severity was evaluated by a point-counting method at 200 magnification as described before (15). An average of 50 fields was graded for each specimen, and there are three different specimens for both mito-I/R group and vehicle-I/R group. The severity of hepatic I/R injury was graded as follows: grade 0, minimal or no evidence of injury; grade 1, cytoplasmic vacuolation and foal nuclear pyknosis; grade 2, extensive nuclear pyknosis, cytoplasmic eosinophilia, and loss of intercellular border; grade 3, severe necrosis with disintegration of hepatic cord, hemorrhage, and neutrophil infiltration.

Apoptosis assay We used a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to evaluate the cell death after I/R injury (6). Liver sections that were 7 2m thick were used. The labeled cells were detected using a peroxidase streptavidin conjugate followed by diaminobenzidine staining (TdT-FragEL DNA Fragmentation Detection Kit, QIA33; Calbiochem, San Diego, Calif). The apoptotic index was calculated as the percentage of TUNEL-positive cells in one 400 magnifying field. The yields of the apoptotic index in 50 selected fields in each sample were averaged.

Western blot analysis In an attempt to quantify the cytochrome c released from mitochondria and the level of cleaved caspase 9 in the partial liver ischemia model, the cytosolic and mitochondrial portions of the liver samples were separated by differential centrifugation. The protein concentration in each sample was measured with a Bradford protein assays using bovine serum albumin (BSA) as a standard. The collected cytosolic samples (30 2g) were subjected to 15% sodium dodecyl sulfateYpolyacrylamide gel electrophoresis. The samples were incubated with polyclonal rabbit antiYcytochrome c antibody (Cell Signaling, Danvers, Mass) or polyclonal cleaved caspase 9 antibody (Cell Signaling), followed by a horseradish peroxidaseYconjugated secondary antibody. Specific bands were detected with an ECL detection system (Amersham, Sweden), and the protein signals were quantified using computer-assisted densitometry. To quantitate the lipid peroxidation produced in oxidative stress, we measured the amount of 4-hydroxynonenal (4-HNE) modified protein, an !," unsaturated hydroxyalkenal produced by lipid peroxidation, in the partial liver I/R model. Briefly, the liver samples were lysed in lysis buffer containing 50 mM Tris-HCl, 0.25% sodium deoxycholate, 150 mM NaCl, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein lysates were centrifuged at 4-C for 30 min at 14,000g. After centrifugation, the supernatant fluid was collected, and the protein content in each sample was determined with a Bradford protein assay using BSA as a standard. Equal amounts of the proteins (30 2g) were loaded to 15% gels and then separated by polyacrylamide gel electrophoresis. 4-Hydroxynonenal (#46545; Abcam, Cambridge, Mass) (1:1,000 dilution) was probed as a biomarker of oxidative stress. Following exposure to horseradish peroxidaseYconjugated secondary antibodies, the blots were imaged and analyzed as described previously.

Statistical analysis All data were analyzed using SPSS 11.0 (Chicago, Ill). The data are expressed as the mean T SE. A one-way analysis of variance was used for the comparisons of two groups. P G 0.05 was considered to be significant.

RESULTS The integrity and distribution of isolated mitochondria in the liver of recipient rats

The purity of isolated mitochondria was checked by examining the expression of specific mitochondrial marker, COX IV, in the lysate. As shown in Figure 1, all isolated mitochondrial

Serum alanine aminotransferase level The serum alanine aminotransferase (ALT) level was measured before and 240 min after reperfusion in each group (n = 6) to ascertain the I/R injury (6). Alanine aminotransferase activity was determined using a prepared kit (Procedure no. 505; Sigma Diagnostics, St Louis, Mo), according to the instruction manual.

FIG. 1. The purity of the isolated mitochondria. The purity of the isolated mitochondrial lysate was examined by gel electrophoresis. The samples showed only the expression of a specific mitochondrial marker, COX IV, but not the protein of the cytosolic component, "-actin.

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lysate showed only COX IX but not "-actin (cytosolic component) expression. To confirm the integrity and function of the infused mitochondria, we first measured the membrane potential ($=m) of the isolated mitochondria by flow cytometry. As shown in Figure 2A, the isolated mitochondria stained with JC-1 expressed $=m with a high signal of red fluorescence (left panel). The isolated mitochondria treated with CCCP (a mitochondrial membrane potential disrupter) showed decreased red fluorescence (right panel). The isolated mitochondria labeled with MitoTracker Orange CMTMRos (Invitrogen), which labels mitochondria that maintain their membrane potential, were infused via an intrasplenic injection. As an internal control, MitoTracker only was infused in another group of test animals. After 240 min, the liver of the recipient rat was harvested, cryosectioned, and observed using confocal microscopy. As shown in Figure 2B, the MitoTracker OrangeYlabeled mitochondria were distributed among the liver parenchyma. In addition, no staining was found in the liver when only the MitoTracker dye was infused (figure not shown).

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FIG. 3. Effects of isolated mitochondria infusion on the ALT levels. The baseline ALT levels of the four groups were within reference range. After 240-min reperfusion, the increase in the serum ALT level was significantly less in the I/R-mito group than that of the I/R-vehicle group. The values are expressed as the mean T SE; #P G 0.05 compared with the other two groups; *P G 0.05 between the I/R-mito and I/R-vehicle groups (n = 6 in each group).

The effects of infusing isolated mitochondria on the serum ALT level

To validate the protective effects of infused mitochondria, the serum level of ALT was determined before ischemia (basal) and 240 min after reperfusion. As shown in Figure 3, the basal serum levels of ALT in the four groups (sham, shammito, I/R-mito, and I/R-vehicle) were all within reference range (25.7 T 5.2, 27.0 T 4.5, 26.5 T 1.4, and 17.5 T 2.5 U/L; P 9 0.05). After 240 min of reperfusion, a marked elevation of ALT levels was noted in the I/R-vehicle group (414.3 T 67.1 U/Lj1), whereas the I/R-mito group (208.8 T 30.2 U/Lj1) showed less elevation of ALT when compared with those of I/R-vehicle group (P G 0.05). There was only a mild elevation of ALT after 240 min of reperfusion in the sham (65 T 13.8 U/Lj1) and sham-mito (71.3 T 15.9 U/Lj1) groups, probably because of the surgical and anesthetic trauma. Our results indicate that mitochondria infusion attenuated tissue injury after I/R injury in the liver. Besides, because there was no significant difference of ALT levels in the sham and shammito groups, we used data of sham group as a control in the rest of our study.

The effect of mitochondrial infusion on the histopathologic change in the liver

After 240 min of reperfusion, the ischemic lobes of the liver were harvested and sectioned for hematoxylin-eosin staining. As shown in Figure 4, the liver infused with MitoTracker only (I/R-vehicle: MitoTracker Orange infusion group) showed congestion, hemorrhage, and necrosis of the hepatocytes following reperfusion injury. The liver infused with isolated, functional mitochondria (I/R-mito: mitochondria infusion group) showed a mild ballooning change in the cytosolic parts of the liver only. The grading of the severity of liver injury between the two groups was analyzed, and it showed that I/R-mito group has less tissue injuries (P G 0.012). The effect of mitochondrial infusion on cell deaths in the liver

To evaluate cell deaths after reperfusion, we performed TUNEL staining on the paraffin sections of the liver. As shown in Figure 5, after 240-min reperfusion, the average

FIG. 2. The integrity and biodistribution of infused mitochondria. A, Flow cytometry analysis of the mitochondrial membrane potential ($=m) of the isolated mitochondria stained with JC-1.The change in $=m was reported by the ratio of red to green fluorescence. High red fluorescence signals indicated that the membrane potentials of the infused mitochondria were well maintained (left panel). After treating the mitochondria with CCCP (carbonylcyanide m-chlorophenylhydrazone), a protonophore that induces mitochondria permeability transition, the $=m, was disrupted (right panel). B, The mitochondria labeled with MitoTracker Orange were distributed in hepatocytes 240 min after infusion (right panel, 100; left panel, 200; scale bar = 50 2m.).

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FIG. 4. Hematoxylin-eosin staining of the liver after reperfusion injury. After 240-min reperfusion, the liver sections of the I/R-vehicle group showed congestion, hemorrhage, and necrosis of the hepatocytes after reperfusion injury. The liver infused with isolated, functional mitochondria (I/R-mito group) showed mild ballooning change of the cytosolic parts of the liver only (n = 6 in each group). Magnification: left panel, 100; right panel, 200. Scale bar = 50 2m.

apoptotic counts in hepatocytes were 59.5% T 4.4% in the I/R-vehicle group and 24.6% T 9.1% in the I/R-mito group. The results showed that mitochondria infusion decreased cell death after reperfusion injury. The effect of mitochondrial infusion on the expression of cytochrome c and caspase 9 in the liver after reperfusion injury

To assess the effect of mitochondria infusion on cell death in I/R injury, we used Western blot analysis to determine the release of cytochrome c and cleaved caspase 9, markers of the mitochondrial apoptotic pathways, in the cytosolic fraction of hepatocytes experiencing I/R injury. As shown in Figures 6 and 7, the expression of cytochrome c and cleaved caspase 9

in the cytosolic parts of hepatocytes significantly increased after reperfusion injury (I/R-vehicle), when compared with those of sham groups (P G 0.05). The infusion of isolated mitochondria (I/R-mito) effectively mitigated the cytochrome c release and the expression of cleaved caspase 9 from mitochondria in reperfused livers (P G 0.05, when compared with the I/R-vehicle group). The results showed that mitochondria infusion could reduce mitochondrial damage and subsequent cell death after reperfusion insults. The effect of mitochondrial infusion on oxidative stress in hepatocytes after I/R injury

The uncontrolled production of ROS plays an important role in inducing I/R injury in the liver. Nonscavenged ROS cause

FIG. 5. TUNEL staining of the liver after reperfusion injury. A, After 240 min of reperfusion, the liver sections of the I/R-vehicle group showed many TUNEL-positive cells, whereas the livers of the I/R-mito and the sham groups showed fewer stained cells (magnification: left panel, 100; right panel, 200; scale bar = 50 2m). B, The average counts of the TUNEL-positive hepatocytes between the three groups. *P G 0.05 between groups (n = 6 in each group).

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FIG. 6. The effects of mitochondrial infusion on the expression of cytochrome c in the livers after reperfusion injury. Upper panel, representative Western blot; lower panel: densitometry results. P G 0.05 when comparing the I/R-mito and I/R-vehicle groups (*) (n = 6 in each group).

damage to cellular proteins, lipids (lipid peroxidation), and nucleic acids and result in cell death. To determine whether mitochondrial infusion protects against reperfusion-induced oxidative stress in the liver, we measured the product of lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE), in the liver from animals experiencing partial liver I/R with and without the infusion of isolated mitochondrial. 4-Hydroxynonenal is an unsaturated !,"-hydroxyalkenal that is generated during the lipid peroxidation cascade in oxidative stress, and 4-HNEY modified proteins are used as biomarkers of oxidative stress. As shown in Figure 8, after 45-min reperfusion, there was a significant increase in 4-HNEYmodified proteins in the liver,

FIG. 7. The effects of mitochondrial infusion on the expression of cleaved caspase 9 in the livers after reperfusion injury. Upper panel, representative Western blot; lower panel: densitometry results. P G 0.05 when comparing the I/R-mito and I/R-vehicle groups (*) (n = 6 in each group).

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FIG. 8. The effects of mitochondrial infusion on the lipid peroxidation in the livers after reperfusion injury. Upper panel, the representative Western blotting; lower panel: densitometry results. P G 0.05 when comparing the I/R-mito and I/R-vehicle groups (*) (n = 4 in each group).

and the infusion of isolated mitochondrial decreased the expression of 4-HNEYprotein conjugates (P G 0.05). DISCUSSION This study demonstrates the protective effects of a splenic infusion of isolated and functional mitochondria on hepatic I/R injury in a rat model. The work has two main findings. First, the isolated mitochondria from donor animals maintained intact membrane potential in the recipient animal’s liver 240 min after infusion. Second, the infusion of isolated mitochondria mitigated the tissue insults, oxidative stress, and cell death in the livers after reperfusion injury. A growing body of evidence indicates that mitochondrial dysfunction is a critical pathological mechanism in reperfusion injury of the liver. Impaired mitochondria result in defective energy utilization and excessive ROS levels (12). Therefore, targeting the mitochondria is a promising approach for the treatment of liver reperfusion injury. A previous study showed that a selective MPT inhibitor, NIM811, mitigated reperfusion injury after liver transplantation (16). Another report demonstrated that diazoxide, a selective mitochondrial ATPdependent K+ channel agonist, attenuated graft injury after mouse liver transplantation (17). A new study demonstrated that stem cells can repair lipopolysaccharide-injured alveolar cells via the transfer of mitochondria (18). Therefore, it might be rational to transfer fresh mitochondria to the ischemic tissue for rescue. In the previous work, it was identified that mitochondrial injection into ischemic myocardium provides protection against reperfusion injury. We reproduced a similar protective effect of mitochondrial infusion on a different tissue, the liver. We replenished the ischemic liver with isolated, intact mitochondria from donor livers. Our results show that mitochondria infused via the spleen were functional and could attenuate the reperfusion injury of the liver.

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SHOCK MARCH 2013 The first prerequisite of mitochondrial replenishment in our study was to effectively deliver the isolated mitochondria to the endangered hepatocytes. We used the splenic injection because the return of the splenic vein would transport the mitochondria to the liver via the portal system. In addition, the mitochondria (4Y6 nm) are much smaller than the liver endothelial fenestrate (98 T 13.0 nm), which would enhance their distribution within the liver parenchyma (19). Although splenic injection has been classically used for cancer metastasis studies, this delivery method was also shown effective for mitochondrial infusion. Of course, the infusion of isolated mitochondria via other routes, such as peripheral veins, seems to be more reasonable in translational significance. However, the dynamics of delivering mitochondria in peripheral blood would be complicated and requires further studies. The second requisite was for the mitochondria to maintain functionality throughout the reperfusion period. Although we did not examine the oxygen consumption of the respiratory chains in the isolated mitochondria, our study showed that the infused mitochondria maintained their intact membrane potential for at least 240 min after reperfusion began. The third critical determinant of the protective effect is the timing of intervention. Early reperfusion is an important period for salvaging ischemic tissue, as shown in postconditioning studies (20, 21). The rapid production of ROS, the opening of the MPT pore (22), and fluctuations in pH (23) all develop during this period. Although we did not explore the exact mechanisms, our data showed that lipid peroxidation was reduced in the isolated mitochondria-infused livers. We proposed that the infused, respiration-competent mitochondria might supplement a working ROS scavenging system and increased ATP. A decrease in ROS production would reduce the activation of downstream signaling, especially the mitochondria permeability transition. The restored energy would help maintain the electrochemical coupling and calcium homeostasis. Thus, the infusion of mitochondria just before reperfusion has clinical implications for the treatment of acute ischemic diseases. The pathophysiological mechanisms of I/R injury are complex. Researchers have found that the initial phase (within 2 h) following liver I/R is characterized by oxidative stress (24), cytokine activation (25), and MPT (26), whereas the late phase (after 6 h of ischemia) involves the activation of neutrophils, the upregulation of adhesion molecules, and the impairment of microcirculations (27). Although our results show that a mitochondrial infusion is effective in mitigating the initial ischemic injury, the question of whether this treatment could also affect the late phase of injury deserves further study. On the other hand, recent researches identified the mitochondrial DNA as a source of damage-associated molecular patterns (DAMPs) that induced inflammatory response (28). It has been estimated that mitochondrial DAMPs produced from 5% of animal’s liver could induce marked lung injury (29). Therefore, the isolated mitochondria must be kept intact to prevent the production of mitochondrial DAMPs. In summary, our study shows that the infusion of isolated mitochondria at the beginning of the reperfusion period mitigated the cellular injury of liver in a partial liver I/R injury

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model. The cytoprotective mechanisms of the mitochondria infusion on the liver require further study. ACKNOWLEDGMENTS The authors thank P. H. Chan, James R. Doty Professor of Neurosurgery and Neurosciences at Stanford University, for advice in design and techniques in mitochondrial researches.

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